ZOMETA

1
DIAGNOSI E TERAPIA DELLE LEUCEMIE LINFOBLASTICHE
ACUTE e DELLE SINDROMI LINFOPROLIFERATIVE
CRONICHE, DEI LINFOMI E DEL MIELOMA
MULTIPLO Giovanni Martinelli, MD Istituto di
Ematologia e Oncologia Medica L. A. Seràgnoli,
Università degli Studi di Bologna
2
ALL
DANNO GENETICO
ANLL o AML
3
Lanalisi molecolare

• Identifica gruppi di pazienti con diversa
  prognosi
• Anomalie genetiche non citogeneticamente
  identificate o caratterizzate
• Significato clinico diverso della persistenza di
  MMR
• Livelli di malattia minima residua assai piccoli.
• Suggerisce la risposta alla chemioterapia
• Meccanismi molecolari sconosciuti.

4
Geni non di Fusione

• Si definiscono geni non di fusione gli oncogeni
  associati a traslocazioni cromosomiche
  specifiche, generati dalla ricombinazione tra due
  geni.
• Danno origine a m-RNA neoplastici, il cui
  prodotto e spesso over espresso ed e oncogeno
  anche se solo uno dei due geni e funzionante.
• Un tipico esempio e loncogene Myc associato
  alla traslocazione t(814)

Istituto di Ematologia ed Oncologia Medica
Seràgnoli, Bologna
5
GENI NON DI FUSIONE
A
C
C
R
R
C
B
R
6
Geni di Fusione

• Si definiscono geni di fusione gli oncogeni
  associati a traslocazioni cromosomiche
  specifiche, generati dalla ricombinazione tra due
  geni.
• Danno origine a m-RNA chimerici il cui prodotto
  e espresso ed e neoplastico se entrambi i due
  oncogeni sono funzionanti.
• Un tipico esempio e loncogene BCR-ABL associato
  alla traslocazione t(922) ed alla Leucemia
  Mieloide Cronica, oppure loncogene PML-RAR alfa
  associato alla t(1517) ed alla Leucemia Acuta a
  promielociti.

Istituto di Ematologia ed Oncologia Medica
Seràgnoli
7
GENI DI FUSIONE
C
C
R
R
C3
C5
R
8

Acute Lymphoid Leukemia
9
FREQUENCY OF PRINCIPAL TRANSLOCATION AND FUSION
GENEs IN ALL
10
ALL
11
Principali alterazioni molecolari e fusion gene e
loro frequenza nelle ALL

• none 19
• random 25
• BCR-ABL 4
• TEL-AML1 28
• MLL fusions 6
• 11q23 (AMLL1/Htrx/Hrx/ALL-1) 13 (1 )
• E2A-PBX 1

Istituto di Ematologia ed Oncologia Medica
Seragnoli, Bologna
12
Estimated Frequency of Specific Genotypes of ALL
in Children and Adults.
13
Transformation of Hematopoietic Cells in the
Pathogenesis of ALL.
14
Mechanism of Transcriptional Repression by
TEL-AML1.
15
The Retinoblastoma (RB) and p53 Tumor-Suppressor
Network.
16
KaplanMeier Analysis of Event-free Survival
According to the Subtype of Leukemia in 467
Children with ALL Who Were Enrolled in Three
Consecutive Treatment Protocols at St. Jude
Children's Research Hospital from 1991 to 1999.
17
Influence of Host Germ-Line and ALL Blast
Genotypes on the Probability of Cure and of
Adverse Events.
18
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19
Analisi molecolare Microarray
20
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21
Principali alterazioni molecolari e fusion gene e
loro frequenza nelle ALL

• random 25
• BCR-ABL 4
• TEL-AML1 28
• MLL fusions 6
• 11q23 (AMLL1/Htrx/Hrx/ALL-1) 13 (1 )
• E2A-PBX 1
• none 19

Istituto di Ematologia ed Oncologia Medica
Seragnoli, Bologna
22
Is ALL Ph homogenous? INDIVIDUAL VARIABILITY?
23
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24
Struttura dei geni ABL e BCR e sonde utilizzate
in FISH a 3 colori e in D-FISH (Oncor)
Sinclair et al., Blood 2000 95738-744
25
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26
cen
LMX2
NEK6 PSMB7 NR5A1 NR6A1 RPL35 GOLGA1 PPP6C H
SPA5 SIN1_HUMAN PBX3 ANGPTL2 SLCA8 RPL12 STXBP1 SH
2D3C CDK9 FPGS ENG AK1 SIAT7D DPM2 ZNF297B LMX1B C
9orf15 C9orf16 LCN2 CIZ1_HUMAN DNM1 GOLGA2 SLC27A4
ODF2 GLE1L SPTAN1 SET SH3GLB2 CRAT PPP2R4 CCBL1 E
NDOG PMX2_HUMAN PTGES DYT1 USP2O FREQ ASS FUBP3 PR
DM12 RRP4_HUMAN
Prostaglandin E synthase
chr.9
tel
27
CDK9 FPGS ENG AK1 SIAT7D DPM2 ZNF297B LMX1B C9orf1
5 C9orf16 LCN2 CIZ1_HUMAN DNM1 GOLGA2 SLC27A4 ODF2
GLE1L SPTAN1
SET SH3GLB2 CRAT PPP2R4 CCBL1 ENDOG PMX2_HUMAN PTG
ES DYT1 USP2O FREQ ASS FUBP3 PRDM12 RRP4_HUMAN ABL
LMX2
NEK6 PSMB7 NR5A1 NR6A1 RPL35 GOLGA1 PPP6C HS
PA5 SIN1_HUMAN PBX3 ANGPTL2 SLCA8 RPL12 STXBP1 SH2
D3C
28
LMX2
NEK6 PSMB7 NR5A1 NR6A1 RPL35 GOLGA1 PPP6C H
SPA5 SIN1_HUMAN PBX3 ANGPTL2 SLCA8 RPL12 STXBP1 SH
2D3C CDK9 FPGS ENG AK1 SIAT7D DPM2 ZNF297B LMX1B C
9orf15 C9orf16 LCN2 CIZ1_HUMAN DNM1 GOLGA2 SLC27A4
ODF2 GLE1L SPTAN1 SET SH3GLB2 CRAT PPP2R4 CCBL1 E
NDOG PMX2_HUMAN PTGES DYT1 USP2O FREQ ASS FUBP3 PR
DM12 RRP4_HUMAN
LMX2
NEK6 PSMB7 NR5A1 NR6A1 RPL35 GOLGA1 PPP6C H
SPA5 SIN1_HUMAN PBX3 ANGPTL2 SLCA8 RPL12 STXBP1 SH
2D3C CDK9 FPGS ENG AK1 SIAT7D DPM2 ZNF297B LMX1B C
9orf15 C9orf16 LCN2 CIZ1_HUMAN DNM1 GOLGA2 SLC27A4
ODF2 GLE1L SPTAN1 SET SH3GLB2 CRAT PPP2R4 CCBL1 E
NDOG PMX2_HUMAN PTGES DYT1 USP2O FREQ ASS FUBP3 PR
DM12 RRP4_HUMAN
LMX2
NEK6 PSMB7 NR5A1 NR6A1 RPL35 GOLGA1 PPP6C H
SPA5 SIN1_HUMAN PBX3 ANGPTL2 SLCA8 RPL12 STXBP1 SH
2D3C CDK9 FPGS ENG AK1 SIAT7D DPM2 ZNF297B LMX1B C
9orf15 C9orf16 LCN2 CIZ1_HUMAN DNM1 GOLGA2 SLC27A4
ODF2 GLE1L SPTAN1 SET SH3GLB2 CRAT PPP2R4 CCBL1 E
NDOG PMX2_HUMAN PTGES DYT1 USP2O FREQ ASS FUBP3 PR
DM12 RRP4_HUMAN
tel
29
cen
FBXW3 IGLL1 ZNF70 VPREB3 MMP11 SMARCB1 SLC2A11 MI
F GSTT2 DDT GSTT1 CABI_HUMAN GGTLA1
SMARCB1 SWI/SNF related, actin dependent
regulator of chromatin subfamily B member 1
GSTT1Glutathione S-transferase theta 1
tel
chr.22
30
Principali alterazioni molecolari e fusion gene e
loro frequenza nelle ALL

• random 25
• BCR-ABL 4
• TEL-AML1 28
• MLL fusions 6
• 11q23 (AMLL1/Htrx/Hrx/ALL-1) 1
• E2A-PBX 1
• none 19

Istituto di Ematologia ed Oncologia Medica
Seragnoli, Bologna
31
11q23 and MLL
32
MLL positive leukemias
t(1119)
t(111)
t(1122)
t(411)
t(X11) (q13q23)
t(611)
t(X11)(q24q23)
t(911)
MLL
del 11 (q11q23)
t(1011)
del 11 (q21q23)
t(1117)
Breakpoints in MLL
5
6
7
8
10
12
13
9
11
centromere
telomere
Zn fingers
A-T hooks
MT
MLL
fusion point
ENL
MLL
ENL
der 11 chromosome
33
MLL and Fusion Patners in Leukemia

ENL
MLL
AF10
CALM
AFA
AF17
AF6q21
AF5q31
ELL
AF19
AFX
Eps15
ABI
ENN
MSF
AF3q21
AMLL1/Htrx/Hrx/ALL-1
AT
PHD
MTase
BP??
34
Chr. 8 and t(814) and BURKITT LUKEMIA
35
TRANSLOCATIONS OF c-Myc Locus

• Reciprocal chromosomal translocations in
  Burkitt's lymphoma, a solid tumour of B
  lymphocytes involves chr.14 and
• chr 8
• chr.2
• chr 22.

36
The genes for making the heavy chains of
antibodies (CH) are located on chromosomes 14,
whereas those for making the light chains are on
chromosomes 2 and 22.
37
Struttura dellanticorpo
38
Patologia dell sintesi Ig
39
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40
Struttura dellanticorpo

• Le Ig possono essere suddivise in classi o
  sottoclassi sulla base delle catene pesante
• ISOTIPI
• IgM m
• IgA a1 a2
• IgG g1, g2, g3, g4
• IgD d
• IgE e

41
Configurazione geni Ig germinale
IgH Cromosoma 14 Igk Cromosoma 2 Igl
Cromosoma 22
42
Riarrangiamento catena leggera
43
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44
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45
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46
Mapping of cloned germline and rearranged antigen
receptor gene segments
47
Genomic structure of selected antigen receptor
loci
48
Cloning of the V(D)J recombinase genes.
49
The RAG proteins and the RAG cleavage
reaction (A) Schematic of RAG-1 and RAG-2,
illustrating known mofits and the putative RAG-1
DDE active site (B) Summary of the RAG cleavage
reaction.
50
V(D)J recombination and the non-homologous
end-joining pathway
51
(No Transcript)
52
Plasma cell
VKJK
Memory-B
Maturazione del B linfocita
53
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54
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55
Antigene interection with naive B cell

• Secrezione di Ig a bassa affinità
• Partecipazione reazione CG
• Selezione Ag-dipendente
• Proliferazione
• Ipermutazione somatica
• Selezione per gt affinità vs lt affinità e/o
  autoreattività
• Switch isotipico

56
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57
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58
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59
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60
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61
T-cell dependent B-cell immune response
CD40 on B cell CD40L on T cell
(CD154) Proliferazione Survivall Memory
differentiation
62
Activation-Induced Deaminasi (AID)

• Somatic hypermutations (SHM)
• IgV Gene conversion (IGC)
• Class switch recombination (CSR)
• Deaminazione G to U
• G to U
• Repair with Uracil-DNA-glycosylase (UNG)

63
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64
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65
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66
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67
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68
Comparazione delle caratteristiche cliniche e
della sopravvivenza fra i gruppi mutato e
germline.
69
(No Transcript)
70
BASI MOLECOLARI DELLE SINDROMI IMMUNOPROLIFERATIVE
MM
71
MM UN MODELLO DI PATOGENESI
Extra-medullary Myeloma
Intra-medullary Myeloma
Normal Plasma Cell
Myeloma Cell Line
Smouldering Myeloma
MGUS
Primary Ig
translocations
Secondary
(Ig) translocations
Karyotypic instability, 13q/13q14
deletions/monosomy
N-Ras, K-Ras, or FGFR3
mutations
p53 mutations
p16 methylation/ p18 deletion
Istituto Seràgnoli - Bologna
72
14q32
IgH
Istituto Seràgnoli - Bologna
73
14q32
IgH
11q13
BCL-1/ CCND1/ PRAD1, myeov
Istituto Seràgnoli - Bologna
74
14q32
IgH
11q13
4p16
MMSET, FGFR3
BCL-1/ CCND1/ PRAD1, myeov
Istituto Seràgnoli - Bologna
75
14q32
IgH
11q13
4p16
16q23
MMSET, FGFR3
c-maf
BCL-1/ CCND1/ PRAD1, myeov
Istituto Seràgnoli - Bologna
76
14q32
IgH
6p21
11q13
4p16
16q23
CCND3
MMSET, FGFR3
c-maf
BCL-1/ CCND1/ PRAD1, myeov
Istituto Seràgnoli - Bologna
77
14q32
IgH
6p21
11q13
4p16
16q23
CCND3
MMSET, FGFR3
20 of pts
with 14q translocations
c-maf
Istituto Seràgnoli - Bologna
78
14q32
IgH
6p21
11q13
4p16
16q23
CCND3
20 of pts
with 14q translocations
c-maf
10-15 of pts with 14q
translocations
Istituto Seràgnoli - Bologna
79
14q32
IgH
6p21
11q13
4p16
16q23
CCND3
20 of pts
with 14q translocations
10-15 of pts with 14q
translocations
8 of pts with 14q
translocations
Istituto Seràgnoli - Bologna
80
14q32
IgH
6p21
11q13
4p16
16q23
20 of pts
with 14q translocations
4 of pts with 14q
translocations
10-15 of pts with 14q
translocations
8 of pts with 14q
translocations
Istituto Seràgnoli - Bologna
81
IgH locus
oncogene
reg
coding
reg
coding
TRANSLOCATION
IgH locus
oncogene
reg
coding
TRANSCRIPTIONAL UPREGULATION
Istituto Seràgnoli - Bologna
82
MM UN MODELLO DI PATOGENESI
Extra-medullary Myeloma
Intra-medullary Myeloma
Normal Plasma Cell
Myeloma Cell Line
Smouldering Myeloma
MGUS
Primary Ig
translocations
Secondary
(Ig) translocations
Karyotypic instability, 13q/13q14
deletions/monosomy
N-Ras, K-Ras, or FGFR3
mutations
p53 mutations
p16 methylation/ p18 deletion
Istituto Seràgnoli - Bologna
83
MICROARRAY ANALYSIS
Istituto Seràgnoli - Bologna
84
MICROARRAY ANALYSIS
Tutti i pz affetti da MM di nuova diagnosi
overesprimono una delle cicline D
Istituto Seràgnoli - Bologna
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MECCANISMI DI UP-REGOLAZIONE
DELLE CICLINE D NEL MM
t(414)
t(1114)
? (unknown)
t(614)
t(1416)
?Cyc D2
?Cyc D3
?Cyc D1
?
?
?
Multiple Myeloma
Istituto Seràgnoli - Bologna
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LA FAMIGLIA DELLE CICLINE D
CCND1 CCND2 CCND3
Genomic location 11q13 12p13
6p21 Protein size, kD 33
34 33 Expression in
none high low
hematopoietic tissue


Istituto Seràgnoli - Bologna
87
Cell cycle progression
growth factors
P
DP
P
pRB
E2F
HDAC
pRB/E2F-mediated transcriptional repression of
genes required for G1/S transition
early G1
late G1
S
Istituto Seràgnoli - Bologna
88
Cell cycle progression
growth factors
cyc D
P
DP
P
pRB
E2F
HDAC
pRB/E2F-mediated transcriptional repression of
genes required for G1/S transition
early G1
late G1
S
Istituto Seràgnoli - Bologna
89
Cell cycle progression
growth factors
cyc D
CDK4/6
P
DP
P
pRB
E2F
HDAC
pRB/E2F-mediated transcriptional repression of
genes required for G1/S transition
early G1
late G1
S
Istituto Seràgnoli - Bologna
90
Cell cycle progression
growth factors
P
cyc D
P
P
P
P
pRB
CDK4/6
P
DP
P
P
pRB
DP
E2F
E2F
HDAC
phosphorilation by cdk4/cyc D and cdk2/cyc E
relieves pRB repression of E2F transcription
factors
pRB/E2F-mediated transcriptional repression of
genes required for G1/S transition
early G1
late G1
S
Istituto Seràgnoli - Bologna
91
Cyclin D1 and CDK inhibitors
92
Cyclin D gene family members
CCND1 CCND2 CCND3
Genomic location 11q13 12p13
6p21 Protein size, kD 33
34 33 Expression
in none low
high
hematopoietic tissue

Istituto di Ematologia e Oncologia Medica
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93
CDC25
CAK
2. dephosphorylation
3. phosphorylation
Tyr15
Thr161
Thr14
cdk4/6
GROWTH FACTORS
cyclin D1
1. cyclin D1 sinthesis and association
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
94
Passage of the Restriction point
P
D
P
P
CDK4
P
pRB
P
E
DP
P
P
pRB
DP
E2F
CDK2
E2F
HDAC
phosphorilation by cdk4/cyc D and cdk2/cyc E
relieves pRB repression of E2F transcription
factors
pRB/E2F-mediated transcriptional repression of
genes required for G1/S transition
early G1
late G1
S
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
95
RAS


Raf 1
PI3K


Ral-GDS
MEK/MAPK
Akt/PKB
-

GSK3b
ERK/MAP
CYCLIN D1



b-catenin TCF
FAK

GSK3b
-
integrins
Wnt
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
96
CCND1/BCL-1/PRAD1 gene
alternative splicing
A/G polymorphism at the splice donor region
(nt.870)
if nt.870 is A preferentially this splicing
pattern
Ex.5
Ex.1 Ex.2 Ex.3
Ex.4
if nt.870 is G preferentially this splicing
pattern
Intron 4
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
97
Cyclin D1 isoforms
Transcript a
Ex.1 Ex.2 Ex.3
Ex.4 Ex.5 3 UTR
cyclin box (nt.312-630)
destruction box (PEST- rich
region)
Ex.1 Ex.2 Ex.3
Ex.4 In.4 3 UTR
Transcript b
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
98
TRASLOCAZIONE t(1114)(q13q32)
q p
Cromosoma 11
BCL-1/CCND1/PRAD1
Cromosoma 14
q p
IgH

switch region
BCL-1
IgH locus
tel.
cen.
Ea
Chr.11
Chr.14
Istituto Seràgnoli - Bologna
99
t(1114), OVERESPRESSIONE CICLINA D1

SIGNIFICATO PROGNOSTICO?
Istituto Seràgnoli - Bologna
100
Fonseca et al. Multiple myeloma and the
translocation t(1114)(q13q32) a report on 13
cases Br J Haematol, 1998 Sonoki et al.
Expression of PRAD1/cyclin D1 in plasma cell
malignancy incidence and prognostic aspects Br
J Haematol, 1998 Hoechtlen-Vollmar et al.
Amplification of cyclin D1 in multiple myeloma
clinical and prognostic relevance Br J Haematol,
1998 Pruneri et al. Immunohistochemical analysis
of cyclin D1 shows deregulated expression in
multiple myeloma with the t(1114) Am. J.
Pathol. 2000
FATTORE PROGNOSTICO SFAVOREVOLE??
101
Fonseca et al. Myeloma and the
t(1114)(q13q32) evidence for a biologically
defined unique subset of patients Blood.
2002993735-41

• DISEGNO DELLO STUDIO
• 336 pz. (ECOG 9486/9487) trattati con
  chemioterapia convenzionale, analizzati in FISH
  per la presenza della traslocazione
  t(1114)(q13q32)

• RISULTATI
• - 53/336 pz. (16) con evidenza di t(1114)
• - pts positivi per t(1114) sopravvivenza più
  lunga
• OS 49.6 vs. 38.7 mesi (log-rank P gt .2)
• PFS 33.0 vs. 27.1 mesi (log-rank P gt .2)

Istituto Seràgnoli - Bologna
102
Moreau et al. Recurrent 14q32 translocations
determine the prognosis of multiple myeloma,
especially in patients receiving intensive
chemotherapy Blood. 20021001579-83

• DISEGNO DELLO STUDIO
• 168 pz. (IFM) trattati con diversi protocolli di
  chemioterapia ad alte dosi, analizzati in FISH
  per le principali traslocazioni 14q32

• RISULTATI
• - 26/168 pz. (15.5) con evidenza di
  t(1114)(q13q32)
• - OS attesa a 80 mesi significativamente più
  lunga per i pazienti t(1114)-positivi (87.5 vs.
  55.4 mesi P0.055)

Istituto Seràgnoli - Bologna
103
Soverini et al. Cyclin D1 overexpression is a
favorable prognostic variable for newly diagnosed
multiple myeloma patients treated with high-dose
chemotherapy and single or double autologous
transplantation Blood, 20031021588-94

• SCOPO DELLA RICERCA
• Analizzare loverespressione della ciclina D1 in
  termini di
• - frequenza
• - correlazione con alterazioni chr.11
• - associazione con delezione/monosomia chr.13
  (D13)
• - significato clinico e prognostico.

Istituto Seràgnoli - Bologna
104

• PAZIENTI E METODI

BM da 74 pazienti affetti da MM di nuova
diagnosi ed arruolati nel protocollo
Bologna 96
quantificazione dellmRNA per ciclina D1
mediante real-time RT-PCR (74
pts)
citogenetica convenzionale (CC) ed eventualmente
FISH per chr. 11 e 13
(46/74 pts)
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105
CORRELAZIONE TRA LIVELLI DI CICLINA D1
E ALTERAZIONI DEL CHR.11
No 11q abn.
t(1114)
11
Normal
(n 28)
(n 9)
(n 9)
(n 10)
Istituto Seràgnoli - Bologna
106
CORRELAZIONE TRA OVERESPRESSIONE
DELLA CICLINA D1 E D13
D13
38 dei pazienti ciclina D1-pos
41 dei pazienti ciclina D1-neg
nessuna associazione
Istituto Seràgnoli - Bologna
107
CORRELAZIONE TRA OVERESPRESSIONE DELLA CICLINA D1
E CARATTERISTICHE CLINICO-LABORATORISTICHE
ALLESORDIO
p
Cyc D1 pos
Cyc D1 neg
32 (43)
42
No. patients
Median age
55
51.5
n.s.
Sex (MF)
2111
n.s.
3111
Durie and Salmon stage
I
3
11
II
4
8
n.s.
III
25
23
Istituto Seràgnoli - Bologna
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p
Cyc D1 pos
Cyc D1 neg
32 (43)
42
No. patients
M component
17
26
IgA
n.s.
IgG
8
10
6
7
BJ
Light chain
k
20
28
n.s.
l
14
12
Median M component concentration
IgA (g/dL)(range)
3.90 (1.00-11.17)
4.20 (2.55-8.40)
IgG (g/dL)(range)
3.80 (2.20-4.20)
n.s.
3.65 (2.56-7.80)
BJ (g/24hrs)(range)
4.25 (1.90-18.60)
3.40 (1.90-6.40)
Istituto Seràgnoli - Bologna
109
p
Cyc D1 pos
Cyc D1 neg
No. patients
32 (43)
42
median BMPC ()(range)
n.s.
40 (10-100)
50 (10-100)
median b2-m (mg/L)(range)
2.20 (1.10-6.90)
2.80 (1.20-16.00)
n.s.
median CRP (mg/L)(range)
3.10 (0.10-70.20)
3.60 (0.10-62.50)
n.s.
median creat (mg/dL)(range)
1.10 (0.70-3.00)
n.s.
0.90 (0.70-1.40)
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110
OVERALL SURVIVAL
1.0
0.9
0.8
0.7
0.6
Cyc D1 pos
Probability of survival ()
0.5
0.4
Cyc D1 neg
0.3
0.2
0.1
6
12
18
24
30
36
42
48
54
0
months
p
Cyc D1 pos
Cyc D1 neg
not reached after 48
43
Median OS
0.6
Istituto Seràgnoli - Bologna
111
TIME TO DISEASE PROGRESSION
1.0
0.9
Cyc D1 neg
0.8
0.7
0.6
Cyc D1 pos
Probability of progression ()
0.5
0.4
0.3
0.2
0.1
6
12
18
24
30
36
42
48
54
0
months
p
Cyc D1 neg
Cyc D1 pos
Median TTP
41
26
0.02
Istituto Seràgnoli - Bologna
112
EVENT-FREE SURVIVAL
1.0
0.9
0.8
0.7
0.6
Probability of survival ()
0.5
Cyc D1 pos
0.4
0.3
0.2
Cyc D1 neg
0.1
54
6
12
18
24
30
36
42
48
0
months
p
Cyc D1 neg
Cyc D1 pos
Median EFS
33
24
0.055
Istituto Seràgnoli - Bologna
113
CICLINA D1 vs. D13
nei 18 pts con D13
D13 pos/Cyc D1 neg (n9)
D13 pos/Cyc D1 pos (n9)
26
Median TTP
41
D13 pos/Cyc D1 neg (n9)
D13 pos/Cyc D1 pos (n9)
Median EFS
41
23
Istituto Seràgnoli - Bologna
114
CONCLUSIONI (I)
Loverespressione della ciclina D1

• è unalterazione frequente (43) nei pazienti
  affetti da MM alla diagnosi

• è strettamente correlata ad alterazioni del
  cromosoma 11
• - t(1114),
• - trisomia 11.

Istituto Seràgnoli - Bologna
115
CONCLUSIONI (II)
Loverespressione della ciclina D1

• E un fattore prognostico favorevole
  identifica un sottogruppo di pazienti
  che presentano TTP ed EFS più lunghi
  in seguito a chemioterapia ad alte dosi
  ed autotrapianto.

Istituto Seràgnoli - Bologna
116
QUESITI APERTI
Loverespressione della ciclina D1 è in grado di
controbilanciare la monosomia/delezione del
chr.13?
Istituto Seràgnoli - Bologna
117
t(414)(p16q32)
- cariotipicamente silente - descritta solo nel
MM e MGUS - frequenza nel MM 20ca.
Istituto Seràgnoli - Bologna
118
(No Transcript)
119
t(414) translocation in Multiple Myeloma (MM)
Region 14q32 locus IgH
MM
S Class Switch Recombination (CSR)
Like many tumors of the B-cell lineage, multiple
myeloma (MM) shows recurrent rearrangements of
the immunoglobulin heavy-chain (IGH) locus at
14q32 and molecular studies have identified
FGFR3, CCND1, CCND3, and MAF genes as targets of
primary translocations in this malignancy.
120
unico caso, nel MM, in cui si forma
un gene di fusione (IgH-MMSET),
che rende la t(414)
rilevabile mediante RT-PCR
Istituto Seràgnoli - Bologna
121
Translocations involving locus IgH are observed
in 60-80 of patients with MM
4p16
11q13
16q23
cr.14q32
6p25
6p21

• In patients with MM a number of recurrent
  translocations involving chromosome 14 at band
  q32 have been recently identified, the most
  common being the t(1114) and the t(414).
• Both these chromosomal abnormalities may help to
  identify patients at different risk of death
• The t(414) has been recently reported to be
  associated with an unfavorable clinical outcome.

122
4p16 Region
The t(414) affects at least two potential
oncogenes, MMSET on der(4) and Fibroblast Growth
Factor Receptor 3 (FGFR3) on der (14) the role
of both these genes in the pathogenesis of MM has
not bee fully elucidated.
123
MMSET Multiple Myeloma SET domain protein
Type I
Polyadenilation Segnals
ORF 1911bp 647aa
5UT
3 4 5 6
7 8 9 10
11
N
C
Type II
ORF 4094bp 1365aa
5UT
3 4 5 6
7 8 9 10 12 13 14 15
16 17 18 19 20 21 22 23 24
M.Chesi et al., Blood (1998)
124
MMSET/IgH fusion gene and alternative spicing
IgH 3 4 5 6
MMSET
IgH 4 5 6 MMSET
IgH 5 6 MMSET
125
Region 14q32 locus IgH
MM
S sequenze di DNA ripetitivo, non codificante,
associate alla Class Switch Recombination (CSR)
TRANSLOCATION CSR abherrant OR
mechanismo involving regions near S
126
4p16 Region genic cluster
cr.4p16
127
MMSET Multiple Myeloma SET domain protein
Type I
Polyadenilation Segnals
ORF 1911bp 647aa
5UT
3 4 5 6
7 8 9 10
11
N
C
Type II
ORF 4094bp 1365aa
5UT
3 4 5 6
7 8 9 10 12 13 14 15
16 17 18 19 20 21 22 23 24
M.Chesi et al., Blood (1998)
128
MMSET FUNCTIONALS DOMAIN
N
C
129
QUINDI
1. domini tipici di proteine nucleari coinvolte
in - rimodellamento della cromatina -
meccanismo regolatorio epigenetico
2. deleto nella Wolf-Hirshhorn Syndrome (severi
difetti di crescita, ritardo mentale, difetti
scheletrici etc.)
3. overespresso nel MM per effetto della t(414)
MMSET fattore trascrizionale?
130
Derivatives t(414)
der(14)
der(4)
131
AIM
In the present study we investigated the
frequency and the prognostic relevance of the
t(414) in a series of 63 patients with de novo
MM, who were randomized to receive either a
single autotransplant (Tx-1) or double
autotransplants (Tx-2) as primary therapy for
their disease.

• - frequency and prognostic signifiance t(414)
• in our casistic
• molecular variability of t(414)

• For this purpose we analyzed
• the presence of t(414) by RT-PCR of the hybrid
  transcript between MMSET and the IgH locus
• the overexpression of FGFR3 by Real-time RT-PCR
• the frequency of potentially activating point
  mutations in the FGFR3 translocated coding
  region, by direct sequencing of RT-PCR products.
• The frequency of del 13 and t(414)

132
PATIENTS METHOD

• RT-nested PCR per IgH/MMSET (diagnosis LFU)
• Real-Time PCR per FGFR3 (diagnosis)
• RT-PCR direct sequencing for FGFR3 point
  mutation (diagnosis LFU)

133
Sex (M/F) 12/5 28/18 Age (years, median,
range) 51 (42-61) 55 (40-62) Durie-Salmon
stage (no, ) I 4 (24) 8 (17) II 4
(24) 5 (11) III 9 (52) 33 (72) Isotyp
(no, ) IgG 11 (65) 26 (65) IgA 6
(35) 10 (22) Bence Jones 0 10
(22) High chain (no., ) k 7 (41) 32
(70) l 10 (59) 14 (30) Lytic bone disease
(no, ) 0 7 (41) 9 (20) 1 2 (12) 4
(9) 2 2 (12) 3 (6) gt3 6 (35) 30
(65) Calcium (mg/dl) 9.1 (8.2-12-3) 9.1
(5-13) Hb (g/dl) 11.4 (6.4-14.5) 12.3
(5.6-15-5) Plts 211 (157-300) 212 (59-416) B2
mycroglobulin (mg/L) 2.1 (1.2-6.9) 2.4
(1.1-16) C reactive protein (mg/L) 0.38
(0.01-6.6) 0.31 (0.01-70) Creatinine (mg/dl)
1.1 (0.3-3.0) 1.0 (0.6-1.7) Plasmacells ()
50 (10-90) 50 (10-100)
134
THERAPY RECEIVED
No Tx 0 3 (7) Tx1 11 (65) 26
(56) Tx2 6 (35) 17 (37)
135
RESPONSE TO TRANSPLANT
N. Evaluables 17/17 43/46 Progression
0 1 (2) No response 2 (12) 5
(12) Partial response 11 (65) 15
(35) Complete response (IF) 3 (17) 7
(16) Complete response (IF-) (?) (6) 15
(35)
136
GLOBAL SURVIVAL
n.s
Median Follow-up (months) MMSET/IgH 36
(16-50) MMSET/IgH- 45 (13-72)
137
EVENT FREE SURVIVAL
MMSET/IgH-
p 0.01
MMSET/IgH
Median, range (months) MMSET/IgH 23
(6-29) MMSET/IgH- 32 (6-72)
138
TIME TO DISEASE PROGRESSION in MMSET patients
Median, range (months) MMSET/IgH 23
(6-29) MMSET/IgH- 32 (6-72)
139
t (414) PROGNOSTIC SIGNIFICANCE
- Moreau et al. (Blood, 2002) FISH -
CD138 168 pz. (high dose chemotherapy ) 13
t(414) OS EFS less than (plt0.0001) -
Fonseca et al. (Blood, 2003) FISH - BM
(PC) 351 pts. (conventional chemotherapy ) 13
t(414) OS PFS less than (plt0.001)
t(414) ASSOCIATED TO BAD THERAPY
140
t(414) del(13)
del (13) 5 (42) 7 (29) NO del (13) 7
(54) 17 (71)
141
t(414) del (13)
by 3 years (number percentual)
142
CONCLUSIONS-1

• 27 of patients studied has t(414)
• t(414) worst rensponse to transplant
• EFS TTP shorter in t(414) carring patients

143
t(414) longitudinal study MMSET type of
transcript is conserved through the therapy
144
t(414) longitudinal study alternative spiced
MMSET transcript are associated with longest
clinical outcome
145
t(414)
146
t(414) alternative splicing survival
X 83 alive 47m (28-55m.)
X 45 alive 40m (16-58m.)
147
FGFR3 overexpression point mutations
148
FGFR3 overexpression point mutations
149
FGFR3 overexpression point mutations
150
CONCLUSIONS-2

• t(414) and MMSET was not lost during disease
  progression
• which is the role of aberant transcripts?
• not all the t (414) positive patients
  over-express FGFR3

151
CONCLUSIONS-3

• prognostic significance
• therapeutic implications

152
PROTOCOL MM02
- patients enrolled 126 (at 15/07/03) -
samples for molecular byology 126 (at
diagnosis) 91 (follow up) -
CD138 95 (at diagnosis) 24(post 4
month)
153
PROTOCOL MM02 t(414)
25 patients at diagnosis
9 patients t(414) (36)
16 patients t(414)-
6 patients del13 2 patients NO 1 patients n.v.
2 patients del13 14 patients NO
154
PROTOCOL MM02 genic expression profile
Studiare contemporaneamente lespressione genica
di migliaia di geni
155
GENIC EXPRESSION PROFILE IN MM
- Zahn et al Global gene expression profiling
of multiple myeloma, monoclonal gammopathy of
undetermined significance, and normal bone marrow
plasma cells. Blood (2002)- Claudio et al.
Molecular compendium of gene expressed in
multiple myeloma Blood (2002) - De Vos et al.
Comparison of gene expression profiling between
mulitple myeloma and normal plasmacells
Oncogene (2002) - Underhill et al. Gene
expression profiling reveals a highly specialized
genetic program of plasma cells Blood
(2003) - Shaughnessy JD and Barlogie B
Interpreting the molecular biology and clinical
behavior of multiple myeloma in the context of
global gene expression profiling Immunol. Rev.
(2003)
156
PROTOCOL MM02 genic expression profile
- CD138 selezione magnetica (95 patients at
diagnosis 24 patients post 4 months) - RNA
extraction - vetri MWG 10K
157
Abstract In patients with MM a number of
recurrent translocations involving chromosome 14
at band q32 have been recently identified, the
most common being the t(1114) and the t(414).
Both these chromosomal abnormalities may help to
identify patients at different risk of death in
particular, the t(1114) predicts for good
prognosis, whereas the t(414) has been recently
reported to be associated with an unfavorable
clinical outcome. The t(414) affects at least
two potential oncogenes, MMSET on der(4) and
Fibroblast Growth Factor Receptor 3 (FGFR3) on
der (14) the role of both these genes in the
pathogenesis of MM has not bee fully elucidated.
In the present study we investigated the
frequency and the prognostic relevance of the
t(414) in a series of 63 patients with de novo
MM, who were randomized to receive either a
single autotransplant (Tx-1) or double
autotransplants (Tx-2) as primary therapy for
their disease. For this purpose we analyzed (1)
the presence of t(414) by RT-PCR of the hybrid
transcript between MMSET and the IgH locus (2)
the overexpression of FGFR3 by Real-time RT-PCR
(3) the frequency of potentially activating point
mutations in the FGFR3 translocated coding
region, by direct sequencing of RT-PCR products.
158
Overall, the t(414) was detected by RT-PCR in
17/63 patients (27) of these 17 patients, 13
had both MMSET/IgH fusion gene and FGFR3
overexpression, while 4 patients had MMSET/IgH
but did not overexpress FGFR3. This finding
further confirms the possible discrepancy between
MMSET/IgH positivity and FGFR3 overexpression.
Comparison between t(414) positive and t(414)
negative patients revealed that both groups were
well balanced with respect to the most common
presenting features of MM. In 36 patients, for
whom material was available, FISH analysis for
the detection of 13q deletion and/or monosomy was
performed. Results showed that t(414) positive
patients were more likely to carry del(13) than
t(414) negative patients (46 vs. 29,
respectively). On an intention-to-treat basis,
the probability of attaining stringently defined
complete remission following either Tx-1 or Tx-2
was significantly lower for t(414) positive
patients in comparison with t(414) negative
patients (6 vs. 35, respectively p 0.05).
With a median follow-up of 40 months, no
difference in overall (OS) was detected between
the two groups. At the opposite, in comparison
with the t(414) negative subgroup, patients
carrying the t(414) had significatly lower event
free survival (EFS) (23 vs 32 months,
respectively p0.01) and duration of remission
(23 vs 32 months, respectively p0.01). In
summary, 27 of our MM patients carried the
t(414). In this cohort of homogeneously treated
patients, the t(414) predicted for lower
response to high-dose therapy. Longer follow-up
is required to assess the influence of this
abnormalities on OS. In a subgroup of six
patients carrying t(414), point mutations were
detected in the FGFR3 coding region, thus
suggesting a possible constitutive FGFR3
activation.
159
t(414)(p16q32)
- kariotipicamente silente - described only in
MM e MGUS - frequency in MM 20ca.
160
A subset of multiple myeloma harboring the
t(414)(p16q32) translocation lacks FGFR3
expression but maintains an IGH/MMSET fusion
transcript Madhumita Santra, Fenghuang Zhan,
Erming Tian, Bart Barlogie, and John Shaughnessy
Jr From the Donna and Donald Lambert Laboratory
of Myeloma Genetics at the Myeloma Institute for
Research and Therapy, University of Arkansas for
Medical Sciences, Little Rock, AR
Like many tumors of the B-cell lineage, multiple
myeloma (MM) shows recurrent rearrangements of
the immunoglobulin heavy-chain (IGH) locus at
14q32 and molecular studies have identified
FGFR3, CCND1, CCND3, and MAF genes as targets of
primary translocations in this malignancy. The
multiple myeloma (MM) specific t(414)(p16q32)
not only results in the activation of FGFR3 but
also the creation of a chimeric fusion transcript
between IGH and MMSET. Given the
transcription-activating nature of 14q32
translocations, microarray profiling of global
gene expression has emerged as a powerful method
for identifying IGH-associated rearrangements in
B-cell malignancies. We have previously used this
strategy to identify a novel 14q32 translocation
involving the cyclin D3 gene (CCND3) as well as
all known 14q32 translocations in MM. Using a
combination of gene expression profiling reverse
transcriptase-polymerase chain reaction (RT-PCR)
and interphase fluorescence in situ hybridization
(FISH) we present evidence that nearly 20 of
newly diagnosed cases of MM harbor the
t(414)(p16q32), and approximately 32 of these
cases, while expressing the IGH/MMSET fusion
transcript, lack FGFR3 expression.
Blood, 2003, Vol.101 pp. 2374-2376
161
t(414) PROGNOSTIC SIGNIFIANCE

• La disponibilità di fattori prognostici
  genetici è fondamentale per
• - identificare sottogruppi di pazienti che
  potrebbero condividere il meccanismo
  patogenetico
• proporre eventuali terapie mirate

162
Aim
- studio molecolare t(414) - frequenza e
significato prognostico t(414) nella nostra
casistica
163
FIBROBLAST GROWTH FACTOR RECEPTORS (FGFR)
famiglia di recettori ad attività
tirosin-chinasica coinvolti in una grande varietà
di processi mitogenici e morfogenici sviluppo
embrionale, angiogenesi, riparazione tissutale..
Istituto Seràgnoli - Bologna
164
FIBROBLAST GROWTH FACTOR RECEPTORS (FGFR)
4 membri FGFR1, FGFR2, FGFR3, FGFR4, con
distribuzione (almeno in parte) tessuto
specifica, in grado di riconoscere con affinità
variabile almeno 9 diversi FGFs.
Istituto Seràgnoli - Bologna
165
FGFR3

• glicoproteina di 125 kDa, espressa
  principalmente a livello del SNC, del polmone,
  del rene e delle cartilagini
• il gene che la codifica è stato isolato nel 1990
  da una libreria di cDNA ottenuta dalla linea
  cellulare K562 (Keegan
  et al, Proc. Natl. Acad. Sci., 1991).

Istituto Seràgnoli - Bologna
166
FGFR3 Fibroblast Growth Factor Receptor 3

• is a tyrosin-kinase receptor
• mutazioni nella linea germinale causano una
  grave forma di nanismo
• espressione assente nel tessuto ematopoietico
  delladulto sano
• in MM
• could be over-expressed
• in alcuni casi sono presenti le stesse mutazioni
  che causano il nanismo

167
FGFR3 GENE AT 4p16.3
16,5 kb
coding region
1 2 6 7 (IIIa) 8
(IIIb) 9 (IIIc) 10 18 19
IIIa secreted form
alternative splicing
IIIb epithelial form
IIIc mesenchimal form
Istituto Seràgnoli - Bologna
168
s-s
s-s
I
I
OUT
Ig-like Domains
s-s
s-s
II
II
s-s
s-s
III
III
cell surface
Trans-Membrane Domain
Split Tyrosine Kinase Domain
IN
Istituto Seràgnoli - Bologna
169
Istituto Seràgnoli - Bologna
170
5. Hypochondroplasia (Asn540Lys)
4. Muenke coronal craniosynostosis (Pro250Arg)
1. Achondroplasia (Gly375Cys Gly380Arg)
FGFR3 mutations
3. Crouzon syndrome with acantosis nigricans
(Ala391Glu)
2. Thanatophoric Dysplasia type I and II
(Tyr373Cys Arg248Cys Lys650Glu
Thr807Cys)
Istituto Seràgnoli - Bologna
171
SUBSEQUENT SELECTION OF SOMATIC MUTATIONS
Ex.7 Ex.10 Ex.13 Ex.15
Ex.19
cod.248 TM domain cod.540 cod.650 cod.807
mutations constitutively activated
FGFR3
Istituto Seràgnoli - Bologna
172
EFFECTS OF
FGFR3 ECTOPIC EXPRESSION
ON MURINE B9 MM CELLS
(Plowright EE et al, Blood 2000 95992-997)
FGFR3
phosphorilation
STAT3
P
?
Bcl-xL
ENHANCED PROLIFERATIVE RESPONSE TO IL-6
DECREASED APOPTOSIS
Istituto Seràgnoli - Bologna
173
In multiple myeloma, t(414)(p16q32) is an
adverse prognostic factor irrespective of FGFR3
expression. Keats et al. Blood 20031011520-29
Istituto Seràgnoli - Bologna
174
Clinical and biologic implications of recurrent
genomic aberrations in myeloma Fonseca, et al
Blood 20031014569-4575
175
Recurrent 14q32 translocations determine the
prognosis of multiple myeloma, especially in
patients receiving intensive chemotherapy
Moreau et al. Blood 20021001579-1583
176
Istituto Seràgnoli - Bologna
177
Chr. 12 and t(1221)

• Tel-AML1

178
Chr. 1 and t(119)

• E2a- PBX

179
The genes for making the heavy chains of
antibodies (CH) are located on chromosomes 14,
whereas those for making the light chains are on
chromosomes 2 and 22. These genes are expressed
exclusively in B lymphocytes, because only these
cells have the necessary transcription factors to
switch on their expression. In most (over 90) of
Burkitt's lymphoma cases, a reciprocal
translocation moves the proto-oncogene c-myc from
its normal position on chromosome 8 to a location
close to the antibody heavy-chain genes on
chromosome 14. In other cases, c-myc is
translocated close to the antibody genes on
chromosome 2 or 22. In every case, c-myc now
finds itself in a region of active gene
transcription, and it may simply be the
overproduction of the c-myc product (a
transcription factor essential for cell division)
that propels the lymphocyte down the pathway
towards cancer.
180
Virus and Lymphoma
181
(No Transcript)
182
Figure 1  Schematic representation of the HCV
genome and encoded viral proteins.   The boxed
area corresponds to the single open reading frame
of the hepatitis C virus (HCV) genome. The
stemloop structures represent the 5' and 3'
non-translated (NTR) regions, including the
internal ribosome-entry site (IRES) and 3'X
regions. The function and molecular mass (in kDa)
of the gene products after polyprotein processing
are shown. Core (C)E1, E1E2, E2p7 and
p7non-structural protein 2 (NS2) junctions are
cleaved by a cellular signal peptidase(s) to
yield structural proteins. The NS2NS3
metalloproteinase undergoes autocatalytic
cleavage, which releases the mature NS3 serine
protease. NS3 cleaves the remainder of the NS
polypeptide. The two regions that have extreme
sequence variability in E2, known as
hypervariable regions 1 and 2 (HVR1 and HVR2),
are indicated. A region in NS5A, known as the
interferon (IFN)-sensitivity-determining region
(ISDR), has been linked to the response to IFN-
therapy in some strains of HCV. Both NS5A and E2
have been implicated as antagonists of IFN (for
review, see Ref. 34). ARFP/F, alternative
reading-frame protein/frameshift protein LDLR,
low-density lipoprotein receptor RdRp,
RNA-dependent RNA polymerase.
183
(No Transcript)
184
Cenni di terapia delle ALL nuovi approcci
185
CICLO A1
PREDNISONE
60 mg/sqm p os dal 1 al 5
200 mg/sqm e.v. dal 1 al 5
ENDOXAN
Giorno 6 PAUSA
RITUXIMAB
375 mg/sqm e.v. g 7 (CD20 gt 20)
10 mg/sqm p. os. dal 8 al 12 500 mg/sqm p.c.
g 8 400 mg/sqm ev dal 8 al 12 120 mg/sqm ev
gg 11 e 12 60 mg/sqm ev gg 11 e 12
DEX MTX IFOSFAMIDE ARA-C VM-26
Rachicentesi gg 8 (MTX 12 mg) G-CSF 5 mcgr/Kg
sc dal 14 a PMN gt 1000/mmc
186
CICLO B1 (DAL 28)
RITUXIMAB
375 mg/sqm e.v. g 28 (CD20gt 20)
10 mg/sqm p. os. dal 29 al 33 1 mg ev g
29 500 mg/sqm p.c. g 29 200 mg/sqm ev dal 29
al 33 25 mg/sqm ev gg 32 e 33
DEX VCR MTX ENDOXAN ADRIAMICINA
Rachicentesi gg 29 ( MTX 12 mg) G-CSF 5
mcgr/Kg sc dal 35 a PMN gt 1000/mmc
187
CICLO A2 (DAL 49)
RITUXIMAB
375 mg/sqm e.v. g 49 (CD20gt 20)
10 mg/sqm p. os. dal 50 al 54 500 mg/sqm p.c.
g 50 400 mg/sqm ev dal g 50 al g 54 120
mg/sqm ev gg 53 e 54 60 mg/sqm ev gg 81 e 82
DEX MTX IFOSFAMIDE ARA-C VM-26
G-CSF 5 mcgr/Kg sc dal 56 a PMN gt
1000/mmc Rachicentesi gg 50 (MTX 12 mg)
188
CICLO B2 (DAL 77)
RITUXIMAB
375 mg/sqm e.v. g 77 (CD20gt 20)
10 mg/sqm p. os. dal 78 al 82 1 mg ev g 78
500 mg/sqm p.c. g 78 200 mg/sqm ev dal g 78
al g 82 25 mg/sqm ev gg 81 e 82
DEX VCR MTX ENDOXAN ADRIAMICINA
G-CSF 5 mcgr/Kg sc dal 84 a PMN gt
1000/mmc Rachicentesi gg 78 (MTX 12 mg)
189
CICLO A3 (DAL 98)
RITUXIMAB
375 mg/sqm e.v. g 98 (CD20gt 20)
10 mg/sqm p. os. dal 99 al 103 500 mg/sqm
p.c. g 99 400 mg/sqm ev dal g 99 al g
103 120 mg/sqm ev gg 102 e 103 60 mg/sqm ev
gg 102 e 103
DEX MTX IFOSFAMIDE ARA-C VM-26
G-CSF 5 mcgr/Kg sc dal 105 a PMN gt
1000/mmc Rachicentesi gg 99 (MTX 12 mg)
190
CICLO B3 (DAL 119)
RITUXIMAB
375 mg/sqm e.v. g 119 (CD20 gt20)
10 mg/sqm p. os. dal 120 al 124 1 mg ev g 120
500 mg/sqm p.c. g 120 200 mg/sqm ev dal g
120 al g 124 25 mg/sqm ev gg 123 e 124
DEX VCR MTX ENDOXAN ADRIAMICINA
G-CSF 5 mcgr/Kg sc dal 126 a PMN gt
1000/mmc Rachicentesi gg 120 (MTX 12 mg)
191
CONSOLIDAMENTO
SETTIMANA 21 (G 147) E 24 (G 168)
RITUXIMAB
375 mg/sqm e.v.
RADIOTERAPIA (dopo la fine della chemioterapia)
SNC 24 Gy Bulky mediastinico (gt 7.5 cm) 36
Gy Malattia residua dopo CHT 36
Gy Localizzazioni extranodali decisione libera
192
RAS inhibitors
193
Extracellular signals
RAS GDP (inactive)
Inactivation
Activation
GTPase activating proteins (GAPs)
Exchange factors (GEFs)
RAS GTP (active)
RAS effectors PI3K, MAPK...
Effects cellular growth, proliferation,
differentiation
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
194
Post-translational RAS modifications
sc
Ras
Ras
palmitoyl-PP
CH3
C
fully activated RAS
4. Palmitoylation
farnesyl
S
3. Methylation
Ras
Ras
CAAX
farnesyl
farnesyl-PP
C
S
SH
Farnesyl transferase
Ras
CAAX-protease
2. Proteolysis
C AAX
1. Isoprenylation
farnesyl
S
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
195
Posttranscriptional modification of Ras
P
P
cell membrane
F(or GG)
Ras
4. Palmytolation
C-S
SCH 66336 PHASE II R 115777 PHASE II
Me
Ras
C-S-F(or GG)
PPMTase
SAM
CaaX
3. Methylation
aaX
Ras
Ras
C-S-F(or GG)
Ras
C-S-F(or GG)
FTase Ior GGTase
2.Proteolysis
aaX
1. Prenylation
FPP GGPP
microsomal membrane
196
RAS
PI 3- kinase
RalGDS
Raf
PDK
Rac
Mek
PKB/Akt
Rho
?
Caspase-9
Erk
Bad
GSK3b
?
Forkhead
Ets
?
Fas ligand
p21
Fos
Cyclin D1
Istituto di Ematologia e Oncologia Medica
Seràgnoli - Bologna
197
mTOR inhibitors
198
translation
nutrient transporter
transcription
turnover
tRNA and
TOR
ribosome
autophagy
biogenesis
actin organization
stationary
phase (G0)
Protein Kinase C
signaling
TOR controls a Large and Diverse Set of
Growth-related Readout Green arrows indicate
activation red bars indicate repressionShown is
a composite of yeast and mammalian readouts.
199
RAPAMICIN
RADICICOL
200
TRAIL inhibitors
201

• Targeting the
• Tyrosine kinase act